Alcohol abuse and alcoholism are significant public health problems. In the United States, alcoholism affects approximately 14 million people at a health care cost of $184 billion per year. Human genetic studies on alcoholism have been challenging due to difficulties in quantifying alcohol-related phenotypes and obtaining large sample sizes together with co-morbidity of alcoholism with other behavioral and neuropsychiatric disorders, gender effects, population admixture, and the diversity of mechanisms of vulnerability and resilience to alcohol. The results of many studies indicate that, in addition to a few common alleles, many different rare alleles may contribute to vulnerability in different populations, suggesting a complex genetic architecture that underlies alcohol-related phenotypes. Understanding this genetic architecture requires an integrative systems genetics approach. Drosophila presents an ideal model system, because large numbers of genetically identical individuals can be reared at low cost and without regulatory restrictions, and a plethora of community resources is available for sophisticated genetic manipulations. Previously, we measured ethanol knockdown time in 40 wild-derived inbred Drosophila lines and constructed modules of correlated transcripts associated with alcohol sensitivity and induction of tolerance. In addition, we built computational networks of covariant transcripts around genes that affect sensitivity or resistance to alcohol exposure identified by P-element mutations. During the next phase of our research program we will utilize a novel resource, the Drosophila Genetic Reference Panel (DGRP), that will enable us to capitalize on naturally occurring variation by allowing genome-wide association (GWA) analyses to provide the missing link between DNA polymorphisms and transcriptional network structure, identify eQTL and designate directionality (i.e. cis and trans regulatory effects) to transcriptional networks associated with alcohol induced phenotypes. The DGRP is a community resource, which was generated in our laboratories and consists of 192 wild-derived inbred lines with fully sequenced genomes in which all segregating polymorphisms are known. The lines are genetically variable for all phenotypes measured to date. We will pursue the following Specific Aims: (1) Perform the first GWA study of alcohol sensitivity and tolerance in the 192 DGRP lines to identify polymorphisms associated with alcohol sensitivity and tolerance, use novel statistical models that combine GWA analyses and multiple regressions to generate genotype-phenotype predictions, and validate such predictions by genotyping individual flies from an advanced intercross population derived by crossing DGRP lines with extreme phenotypic values; (2) Combine transcript abundance data from extreme DGRP lines obtained by deep RNA sequencing with GWA information to derive causal genetic networks affecting alcohol sensitivity and tolerance and test network predictions using targeted disruption of hub genes through mutational analysis or RNAi knock-down; (3) Determine to what extent exposure to alcohol during development alters the genetic networks identified in Specific Aims 1 and 2, and identify causal variants associated with genotype by environment interaction for alcohol-related phenotypes. Aim 3 is motivated by our observation that previous chronic alcohol exposure as larvae modulates sensitivity to subsequent alcohol exposures as adults. The proposed studies will result in a comprehensive systems genetics blueprint of the genetic architecture of alcohol sensitivity in Drosophila on which human orthologues can be superimposed to generate novel hypotheses that designate candidate hub genes for future follow-up studies.